Understanding the regulation of beta-cell neogenesis in the pancreas could lead to applications in the field of cell replacement or regeneration therapies for diabetes [Yamaoka T. in Biochem Biophys Res Commun. (2002) 296, 1039-43]. For instance, islet transplantation can restore the functional beta-cell mass in diabetes patients [Shapiro et al in N Engl J Med. (2000)343, 230-238], but it is seriously hampered by the shortage in donor tissue. This problem could be solved by finding ways of generating more islet cells from the available pancreatic tissue, by the process of neogenesis [Bouwens & Kloppel in Virchows Arch. (1996) 427, 553-560]. Despite a lot of progress in the understanding of pancreas development during the last decade, the extracellular factors that specify islet cell differentiation in the embryo remain unknown [Edlund in Nat Rev Genet. (2002) 3, 524-532]. In adult mammals, the endocrine pancreas can expand or regenerate under certain experimental conditions, mainly as a result of islet cell neogenesis from progenitor cells [Bouwens & Kloppel cited supra]. The exact nature of the latter remains elusive while duct cells, acinar cells, or intraislet cells have been suggested bear progenitor capacity [Bouwens & Kloppel in Virchows Arch. (1996) 427, 553-560; Edlund in Nat Rev Genet. (2002) 3, 524-532; Bouwens in Microscopy Research and Technique (1998) 43, 332-336; Guz et al. in Endocrinology. (2001) 142, 4956-4968; Bonner-Weir et al. in Proc Natl Acad Sci USA. (2000) 97, 7999-8004; Ramiya V K et al. Nat Med. (2000) 6, 278-282].
It is difficult to draw firm conclusions from whole pancreas studies both with respect to cell derivation and to the specific regulatory factors. Therefore, in vitro models are preferred to study islet neogenesis starting from defined cell preparations isolated from the pancreas. Very few in vitro studies have been able to demonstrate the feasibility of inducing islet neogenesis from adult tissue. It has already been reported that additional islet cells could be generated from monolayer cultures of adult pancreatic tissue. Confirmation of these findings, in order to unravel the nature of the progenitor cells, of the regulatory factors, and to improve the efficacy of generating islet cells is still lacking.
Human pancreatic derived cell cultures that were treated with KGF (keratinocyte growth factor) and nicotinamide, resulted in increases in insulin content after 3 to 4 weeks. Also, cystic structures containing islet cells budded from the monolayer under influence of extracellular matrix [Bonner-Weir et al. cited supra]. The precursors responsible for this neogenesis were characterised as cells expressing the ductal marker cytokeratin-19 [Gao et al. cited supra]. In another study, long-term cultures were obtained from diabetic NOD mouse pancreas under glucose-free conditions, and these could be stimulated to generate islet-like structures in the presence of glucose [Ramiya V K et al. in Nat Med. (2000) 6, 278-282]. These cells did not reach functional maturity in vitro. It is at present unclear whether the latter observations may have been due to “passenger” stem cells derived from the blood circulation, which have been discovered recently in NOD mice [Kodama et al. in Science (2003) 302, 1223-1227].
Differentiated exocrine cells can revert to a partially dedifferentiated state thereby re-acquiring embryonic plasticity [Bouwens & Kloppel in Virchows Arch. (1996) 427, 553-560; Rooman et al. in Diabetologia 43, 907-914 (2000); Rooman et al. in Gastroenterology 121, 940-949 (2001); Rooman et al. in Diabetes 51, 686-690, (2002)]. This indicated that exocrine cells, the great majority of cells in this organ, can be brought to transdifferentiate into endocrine cells under the appropriate conditions.
Exocrine acinar cells can transdifferentiate into endocrine beta-cells [Bouwens in Microscopy Research and Technique (1998) 43, 332-336] and there have been indications from in vivo studies for the existence of acinar-islet transitional cells [Gu et al. in Pancreas (1997) 15, 246-2501; Bertelli E, Bendayan M in Am J Physiol (1997) 273, C1641-C1649]. Since acinar cells can lose amylase and gain ductal characteristics [Rooman et al. (2000) & (2001) cited supra] the appearance of transitional cells co-expressing ductal markers like cytokeratin and insulin [Wang et al. in Diabetologia. (1995) 38, 1405-1411] could also represent cells that were initially derived from acinar cells. It has been demonstrated that the amylase-secreting cell line AR42J, derived from an acinar tumor, can transdifferentiate into the beta-cell phenotype in vitro [Mashima et al. in J Clin Invest. (1996) 97, 1647-1654]. The present invention reports the in vitro transdifferentiation of acinar cells into beta-cells in a primary culture model with a specific combination of growth factors.
LIF (Leukemia Inhibitory Factor) is a pleiotropic cytokine for which a function in pancreatic development has so far not been described. It is a well-known regulator of stem cell proliferation and differentiation and is widely used to prevent differentiation of embryonic stem cells. Recently, it was reported to stimulate the proliferation of multipotent adult progenitor cells (without differentiation of the cells) in combination with EGF and PDGF [Jiang et al. in Nature (2002) 418, 41-49].
EGF (Epidermal Growth Factor) and other EGF-family members have been implicated in the regulation of embryonic development as well as regeneration of the endocrine pancreas. EGF stimulates proliferation of the undifferentiated pancreatic precursor cells in vitro [Cras-Meneur et al. in Diabetes. (2001) 50, 1571-1579]. In transgenic mice lacking functional EGF-receptors, islet morphogenesis is impaired and beta-cell differentiation is delayed [Miettinen et al. in Development (2000) 127, 2617-2627]. Betacellulin, a growth factor which also operates via the EGF-receptor, was found to promote islet regeneration in subtotally pancreatectomized rats (25Ii) and in alloxan-diabetic mice [Yamamoto et al. in Diabetes. (2000) 49, 2021-2027]. Glp-1, another factor that stimulates beta-cell neogenesis [Drucker in Mol Endocrinol. (2003) 17, 161-171] has also been shown to transactivate the EGF-receptor [Buteau et al. in Diabetes. (2003) 52, 124-132]. In combination with gastrin hormone, EGF was shown to stimulate beta-cell regeneration in streptozotocin-diabetic rats [Brand et al. in Pharmacol Toxicol. (2002) 91, 414-420].
LIF and EGF have been reported to act synergistically as signals that regulate the differentiation of neurons and glial cells in embryos [Viti et al. in J. Neurosci. (2003) 15, 3385-3393]. In astrocyte progenitors EGF increases the competence to interpret LIF as an astrocyte-inducing signal via increased STAT3 phosphorylation. LIF is also considered a key signal for injury-induced neurogenesis in the adult [Bauer et al. in J. Neurosci. (2003) 23, 1792-1803].